Dipeptide alkyl esters and their uses

ABSTRACT

An alkyl ester of dipeptide consisting essentially of natural or synthetic L-amino acids with hydrophobic side chains. Preferable amino acids are leucine, phenylalanine valine, isoleucine, alanine, proline, glycine or aspartic acid beta methyl ester. Preferable dipeptides are L leucyl L-leucine, L-leucyl L-phenylalanine, L-valyl L-phenylalanine, L-leucyl L-isoleucine, L-phenylalanyl L-phenylalanine, L-valyl L-leucine, L-leucyl L-alanine, L-leucine, L-leucyl L-valine, L-phenylalanyl L-valine, L glycyl L-leucine, L-leucyl L-glycine or L-aspartyl beta methyl ester L-phenylalaine. Most preferable dipeptides are L-leucyl L-leucine, L-leucyl L-phenylalanine, L-valyl L-phenylalanine, L-phenylalanyl L-leucine, L-leucyl L-isoleucine, L-phenylalanyl L-phenylalanine and L-valyl L-leucine. 
     The alkyl ester of the dipeptide is most preferably a methyl ester and may also be an ethyl ester or alkyl of up to about four carbon atoms such as propyl, isopropyl, butyl or isobutyl. 
     These alkyl esters of dipeptides consisting essentially of amino acids with hydrophobic side chains may be used to deplete cytotoxic T-lymphocytes or natural killer cells from organisms, cell populations or tissues.

This is a division of U.S. patent application Ser. No. 07/168,177, filedMar. 15, 1988, now U.S. Pat. No. 5,047,401, which is acontinuation-in-part of U.S. Ser. No. 744,051, filed Sep. 9, 1985, nowU.S. Pat. No. 4,752,602.

BACKGROUND OF THE INVENTION

The present invention concerns certain dipeptide esters and their users,particularly for ablation of certain cell-mediated immune responses. Forbrevity and clarity, many of the terms used herein have been abbreviatedand these abbreviations include those shown in Table 1. Researchinvolved in the development of the invention was supported by grantsfrom the United States government.

L-leucine methyl ester (Leu-OMe) has previously been used as alysosomotropic agent (Thiele et al. (1983) J. Immunol. V 131, pp2282-2290; Goldman et al. (1973) J. Biol. Chem. V 254, p 8914). Thegenerally accepted lysosomotropic mechanism involved leu-OMe diffusioninto cells and into lysosomes, followed by intralysosomal hydrolysis toleucine and methanol. The more highly ionically charged leucine, largelyunable to diffuse and of the lysosome, caused osmotic lysosomal swellingand rupture. The fate of leu-OMe subjected to rat liver lysosomes wasadditionally suggested by Goldman et al. (1973) to involve atranspeptidation reaction and a resultant species--"presumably thedipeptide" which was "further hydrolyzed to free amino acids". Asubsequent and related paper by Goldman (FEBS (Fed. Europ. Biol. Sci.)Letters V 33, pp 208-212 (1973)) affirmed that non-methylated dipeptideswere thought to be formed by lysosomes.

L-amino acid methyl esters have been specifically shown to cause ratliver lysosomal amino acid increases (Reeves (1979) J. Biol. Chem. V254, pp 8914-8921). Leucine methyl ester has been shown to cause ratheart lysosomal swelling and loss of integrity (Reeves et al., (1981)Proc. Nat'l. Acad. Sci., V 78, pp 4426-4429).

                  TABLE 1                                                         ______________________________________                                        Abbreviations                                                                                     Symbol                                                    ______________________________________                                        Substance                                                                     L-leucine             leu                                                     L-phenylalanine       phe                                                     L-alanine             ala                                                     L-glycine             gly                                                     L-serine              ser                                                     L-tyrosine            tyr                                                     L-arginine            arg                                                     L-lysine              lys                                                     L-valine              val                                                     L-isoleucine          ile                                                     L-proline             pro                                                     L-glutamic acid       glu                                                     L-aspartic acid       asp                                                     L amino acid methyl esters                                                                          e.g. Leu--OMe                                           L amino acid ethyl esters                                                                           e.g. Leu--OEt                                           D-amino acids         e.g. D-Leu                                              D-amino acids methyl esters                                                                         e.g. D-Leu--OMe                                         dipeptides of L-amino acids                                                                         e.g. Leu--Leu                                           methyl esters of dipeptide                                                                          e.g. Leu--Leu--OMe                                      L amino acids                                                                 cell fraction or type                                                         mononuclear phagocytes                                                                              MP                                                      polymorphonuclear leucocytes                                                                        PMN                                                     natural killer cells  NK                                                      peripheral blood mononuclear cells                                                                  PBM                                                     cytotoxic T-lymphocytes                                                                             CTL                                                     glass or nylon wool adherent cells                                                                  AC                                                      glass or nylon wool non-adherent                                                                    NAC                                                     cells                                                                         Other Materials                                                               phosphate buffered saline                                                                           PBS                                                     thin layer chromatography                                                                           TLC                                                     fluorescence activated cell sorter                                                                  FACS                                                    mixed lymphocyte culture                                                                            MLC                                                     Miscellaneous                                                                 effector:target cell ratio                                                                          E:T                                                     fetal bovine serum    FBS                                                     University of Texas Health                                                                          UTHSCD                                                  Science Center, Dallas, Texas.                                                Standard error of mean                                                                              SEM                                                     probability of significant                                                                          p                                                       difference (Student's t-test)                                                 Graft versus host disease                                                                           GVHD                                                    ______________________________________                                    

Natural killer cells are large granular lymphocytes that spontaneouslylyse tumor cells and virally-infected cells in the absence of any knownsensitization. This cytotoxic activity can be modulated by a host ofpharmacologic agents that appear to act directly on NK effector cells.NK activity has been shown to be augmented after exposure to interferons(Gidlund et al., Nature V 223, p. 259), interleukin 2, (Dempsey, et al.(1982) J. Immunol. V 129, p 1314) (Domzig, et al. (1983) J. Immunol. V130, p 1970), and interleukin 1 (Demsey et al. (1982) J. Immunol. V 129,p 1314), whereas target cell binding is inhibited by cytochalasin B,(Quan, et al. (1982) J. Immunol. V 128, p 1786), dimethyl sulfoxide,2-mercaptoethanol, and magnesium deficiency (Hiserodt, et al. (1982) J.Immunol. V 129 p 2266). Subsequent steps in the lytic process areinhibited by calcium deficiency (Quan et al. (1982) J. Immunol. V 128, p1786, Hiserodt, et al. (1982) J. Immunol. V 129, p 2266), lysosomotropicagents (Verhoef, et al. (1983) J. Immunol. V 131, p 125), prostaglandinE₂ (PGE₂ (Roder, et al. (1979) J. Immunol. V 123, p 2785, Kendall, etal. (1980) J. Immunol. V 125 p 2770), cyclic AMP (Roder, et al. (1979)J. Immunol. V 123, p 2785, Katz (1982) J. Immunol. V 129, p 287),lipomodulin (Hattori, et al. (1983) J. Immunol. V 131, p 662), and byantagonists of lipoxygenase (Seaman (1983) J. Immunol V 131 p 2953).Furthermore, it has been demonstrated that PGE₂ and reactive metabolitesof oxygen produced by monocytes (MP) or polymorphonuclear leukocytes(PMN) can inhibit NK cell function (Koren, et al. (1982) Mol. Immunol. V19, p 1341; and Seaman, et al. (1982) J. Clin. Invest. V 69, p 876).

Previous work by the present applicants has examined the effect ofL-leucine methyl ester on the structure and function of human peripheralblood mononuclear cells (PBM) (Thiele, et al. (1983) J. Immunol. V 131,p 2282.

Human peripheral blood mononuclear cells (PBM) are capable of mediatinga variety of cell-mediated cytotoxic functions. In the absence of anyknown sensitization, spontaneous lysis of tumor cells andvirally-infected cells is mediated by natural killer cells (NK)contained within the large granular lymphocyte fraction of human PBMTimonen et al. (1981) v. J. Exp Med. V 153 pp 569-582. After lymphokineactivation, additional cytotoxic lymphocytes capable of lysing a broadspectrum of tumor cell targets can be generated in in vitro cultures(Seeley et al. (1979) J. Immunol. V 123, p 1303; and Grimm et al. (1982)J. Exp. Med. V 155, p 1823). Furthermore, lymphokine activatedperipheral blood mononuclear phagocytes (MP) are also capable of lysingcertain tumor targets (Kleinerman et al. (1984) J. Immunol. V 133, p 4).Following antigen-specific stimulation, cell mediated lympholysis can bemediated by cytotoxic T lymphocytes (CTL).

While a variety of functional and phenotypic characteristics can be usedto distinguish these various types of cytotoxic effector cells, a numberof surface antigens and functional characteristics are shared. Thus, theantigens identified by the monoclonal antibodies OKT8 (Ortaldo et al.(1981) J. Immunol. V 127, p 2401; and Pertussia et al. (1983) J.Immunol. V 130 p 2133), OKT11 (Pertussia et al. (1983) J. Immunol. V130, p 2133; and Zarling et al. (1981) J. Immunol. V 127, p 2575), NK9(Nieminen et al. (1984) J. Immunol. V 133, p 202) and anti-D44 (Calvo etal. (1984) J. Immunol. V 132, p 2345) are found on both CTL and NK whilethe antigen identified by OKM1 is shared by MP and NK (Zarling et al.(1981) J. Immunol. V 127, p 2575; Ortaldo et al. (1981) J. Immunol. V127, p 2401; Pertussia et al. (1983) J. Immunol. V 130, p 2133; andBreard et al. (1980) J. Immunol. V 124, p 1943. Furthermore, cytolyticactivity of both NK and MP is augmented by interferons, (Kleinerman etal. (1984) J. Immunol. V 133, p 4; Gidlund et al. (1978) Nature V 233, p259; and Trinchieri et al. (1978) J. Exp. Med. V 147, p 1314). Finally,use of metabolic inhibitors has demonstrated some parallels in the lyticmechanism employed by CTL and NK (Quan et al. (1982) J. Immunol. V 128,p 1786; Hiserodt et al. (1982) J. Immunol. V 129, p 1782; Bonavida etal. (1983) Immunol. Rev. V 72, p 119; Podack et al. (1983) Nature V 302,p 442; Dennert et al. (1983) J. Exp. Med. V 157, p 1483; and Burns etal. (1983) Proc. Nat'l. Acad. Sci. V 80, p 7606).

SUMMARY OF THE INVENTION

An alkyl ester of dipeptides consisting essentially of natural orsynthetic L-amino acids with hydrophobic side chains. Preferable aminoacids are leucine, phenylalanine valine, isoleucine, alanine, proline,glycine or aspartic acid beta methyl ester. Preferable dipeptides are Lleucyl L-leucine, L-leucyl L-phenylalanine, L-valyl L-phenylalanine,L-leucyl L-isoleucine, L-phenylalanyl L-phenylalanine, L-valylL-leucine, L-leucyl L-alanine, L-leucine, L-leucyl L-valine,L-phenylalanyl L-valine, L glycyl L-leucine, L-leucyl L-glycine orL-aspartyl beta methyl ester L-phenylalaine. Most preferable dipeptidesare L-leucyl L-leucine, L-leucyl L-phenylalanine, L-valylL-phenylalanine, L-phenylalanyl L-leucine, L-leucyl L-isoleucine,L-phenylalanyl L-phenylalanine and L-valyl L-leucine.

The alkyl ester of the dipeptide is most preferably a methyl ester andmay also be an ethyl ester or alkyl of up to about four carbon atomssuch as propyl, isopropyl, butyl or isobutyl.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that whereas ablation of NK function during incubation withLeu-OMe can be blocked by lysosomotropic agents, there is a productformed during incubation of Leu-OMe with MP or PMN which has effects onNK function no longer blocked by lysosomal inhibitors.

FIG. 2 shows Leu-OMe products of PMN in terms of radioactivity and NKsuppressive effects of TLC fractions.

FIG. 3 shows, the CI mass spectra of TLC fractions with NK toxicactivity and of synthetic Leu-Leu-OMe.

FIG. 4 shows the effects of various agents on losses of NK function fromMP-depleted lymphocytes.

FIG. 5 shows the NK-toxicity of various dipeptide esters.

FIG. 6 shows the loss of NK and MP from PBM incubated with Leu-Leu-OMeat various concentrations.

FIG. 7 shows the toxicity of various Leu-Leu-OMe concentrations forselected cell types.

FIG. 8 shows the Leu-Leu-OMe mediated elimination of precursors ofcytotoxic T lymphocytes, activated NK (A_(c) NK) and NK.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention concerns new compounds and their uses in ablatingparticular cell types and their functions. The presently describedinvention relates to the discovery that dipeptide alkyl esters arecytotoxic metabolites of lysosomotropic amino acid alkyl esters.

It has further been found that alkyl esters of dipeptides consistingessentially of natural or synthetic amino acids with hydrophobic sidechains may function cytotoxically to deactivate natural killer cells(NK) and cytotoxic T lymphocytes (CTL). By the term "hydrophobic" asused herein, is meant uncharged in aqueous solution at physiological pHand also as having no hydroxyl, carboxyl or primary amino groups.

Treatment of NK or CTL with an effective level of an alkyl ester of adipeptide consisting essentially of natural or synthetic amino acidswith hydrophobic side chains serves to deactivate the cytotoxicfunctions of said cells. An effective level varies from circumstance tocircumstance but generally lies between about 25 micromolar and about250 micromolar. An effective level for a whole animal dose generallylies between about 100 mg/kg and about 300 mg/kg.

Both methyl and ethyl esters of dipeptides consisting essentially ofnatural or synthetic amino acids having hydrophobic side chains havebeen specifically found to deactivate natural killer cells or cytotoxicT lymphocytes and other alkyl esters of these dipeptides are confidentlypredicted to have similar or superior effects.

Deactivation of natural killer cells (NK) or CTL cells with suchdipeptide alkyl esters should increase the success of allogeneic bonemarrow transplants by lowering the incidence of graft-versus-hostdisease (GVHD) and by lowering the incidence of transplant rejection.

Graft versus host disease (GVHD) is a major problem in allogeneic bonemarrow transplantation. It occurs in approximately 70% of transplantrecipients and causes death in 20% of those (Wells, et al. p 493 inBasic and Clinical Immunology. Fundenbergo et al. (editors) 2nd ed.Lange, (1978)). The disease occurs when cells of the graft (donor)attack the host tissue, causing abnormalities in the immune system andgastrointestinal tract, as well as skin rashes and liver dysfunction.Although cytotoxic T lymphocytes have traditionally been considered tobe the primary effector cells in GVHD, recent studies have shown acorrelation between the occurrence of the disease and the appearance ofNK activity soon after transplantation. These results implicate thedonor's NK cells in cells in the etiology of GVHD. Moreover, otherstudies demonstrate that high levels of NK activity in a bone marrowrecipient prior to transplantation are associated with GVHD (Dokhelar etal. (1981) Transplantation V 31 p 61; Lopez et al. (1979) Lancet V 2 p1103; and Lopez et al. (1980) Lancet V 2 p 1025). Thus, it is theorizedthat both host and donor NK cells contribute to the development of thedisease.

Current regimens for the prevention and treatment of GVHD consist ofdepleting T-lymphocytes from the donor marrow prior to transplantationand giving the recipient immunosuppressive drugs such ascyclophosphamide and methotrexate, both before and aftertransplantation. The effectiveness of these regimens might be enhancedby treating donor bone marrow and transplant recipients with dipeptidemethyl esters. Potential problems with these therapeutic dipeptide alkylesters and the re-emergence of NK activity from precursors not sensitiveto therapeutic dipeptide alkyl esters.

Currently, bone marrow transplantation is used as a major mode oftherapy in treating aplastic anemia, acute myelogenous leukemia, and avariety of immunodeficiency states. As mentioned above, a majorcomplication of this therapy is graft-versus-host disease (Sullivan etal., Blood V 57, p 207). Severity of GVHD in man correlates withpretransplant levels of natural killer (NK) activity (Lopez et al.,Lancet V 2, p 2101). Thus, by virtue of its ability to diminish NKfunction in vivo, it is contemplated that Leu-Leu-OMe administration,for example, to bone marrow samples prior to their transplantation willbe efficacious in diminishing this complication. An effective level ofthe dipeptide alkyl esters of the present invention for in vitrodeactivation of natural killer cells is between about 10 micromolar andabout 250 micromolar.

Furthermore, in both murine and human models, the incidence of GVHD isdecreased by in vitro treatment of donor bone marrow with agents thatdeplete mature T cells (Korngold et al., Exp. Med. V 148, p 1687;Reisner et al., Blood V 61, 341). Since cytotoxic T cells (CTL) derivedfrom donor bone marrow appear to be the final mediators of GVHD, invitro treatment of donor bone marrow with an agent which selectivelydamages cytotoxic T cell precursors is also likely to be of benefit.Since such an in vitro action of Leu-Leu-OMe has now been demonstratedit is expected that this agent will be of benefit in pre-treating donorbone marrow. An effective level of the dipeptide alkyl esters of thepresent invention for treatment of bone marrow to be transplanted shouldbe between about 10 micromolar and 250 micromolar for ablation ofGVHD-mediating CTL and NK.

A second problem in bone marrow transplantation is the failure ofengraftment (the transplant does not "take" or is rejected). Thisproblem occurs in 10-20% of transplants and can be caused by severalfactors, including improper transplantation technique, extensiveinvasion of the recipient's bone marrow by tumor cells, and rejection ofthe transplant.

The discovery that F¹ mice could reject transplants of parental bonemarrow first indicated that NK cells might be involved in theengraftment failures (Cudkowicz et al. (1971) J. Exp. Med. V 134, p 83;Cudkowicz et al. (1971) J. Exp. Med. V 134, p 1513; and Kiessling et al.(1977) Eur. J. Immunol. V 7, p 655).

Initially graft rejection was thought to be almost totally dependent onT lymphocytes. However, T cells from an F¹ hybrid animal do not normallyattack parental tissue. Therefore, it was suggested that NK cells, not Tcells, mediated the rejection of the parental bone marrow.

Additional support for this hypothesis was derived from the observationthat mice of a strain normally incapable of rejecting bone marrowtransplants acquire this ability when they are injected with cloned NKcells. (Warren et al. (1977) Nature V 300, p 655,). As a result of thesefindings, Herberman et al. ((1981) Science V 214), p 24 have suggestedthat suppression of NK activity might lower the incidence of transplantrejection. This suppression should be achieved by treating the recipientwith the dipeptide methyl esters of the present invention prior totransplantation.

Other clinical uses for alkyl esters of dipeptides consistingessentially of amino acids with hydrophobic side chains, or othersituations where NK or CTL are involved in the pathogeneis of disease.In organ transplants in general (kidney, heart, liver, pancreas, skin,etc.) it is widely accepted that cytotoxic T cells are likely to be thecell type responsible for graft rejection (Mayer et al., J. Immunol. V134, p 258). Thus, it is contemplated that the in vivo administration ofLeu-Leu-OMe or similar dipeptide esters of the present invention will beof benefit in preventing allograft rejection.

It is also contemplated that Leu-Leu-OMe or other alkyl dipeptide estersmay be of benefit in other spontaneously occurring disease states. Avariety of diseases have been classified as "autoimmune diseases"because of the widely accepted belief that they are caused by disordersin the immune system which cause immunologic damage to "self". Thus, ina variety of diseases, including primary biliary cirrhosis, systemiclupus erythematosus, rheumatoid arthritis, multiple sclerosis,autoimmune hemolytic anemia, etc., various forms of immunologic damageto selected organs occur. In some of these diseases, such as primarybiliary cirrhosis, the histologic abnormalities which occur (in thiscase in the liver) closely resemble those which occur in GVHD or inrejection of a transplanted liver (Fennel, (1981), Pathol. Annu. V 16 p289. Thus, it is reasonable that similar mechanisms of cytotoxiclymphocyte damage to liver cells may be occurring, and therefore benefitfrom therapy with Leu-Leu-OMe or other dipeptide alkyl esters of thepresent invention should also occur in such disease states.

The dipeptide alkyl esters of the present invention should be usablechemotherapeutic agents in patients with natural killer cell tumors(generally leukemias), although very few reports of these tumors arefound in the literature (Komiyama et al. (1982) Blood V 60, p 1428(1982); Itoh et al. (1983) Blood V 61, p 940; Komiyama et al. (1984)Cancer V 54 p 1547.

It is contemplated that the dipeptide alkyl esters of the presentinvention may also be used to treat patients with aplastic anemia andother types of bone marrow dysfunction. This suggestion is based onthree sets of observations in human studies: first, NK cells can killnormal bone marrow cells (Hansson, et al. (1981) Eur. J. Immunol. V 11,p 8); second NK cells inhibit growth of blood cell precursors in vitro(Hansson, et al. (1982) J. Immunol. V 129, p 126; Spitzer et al.: BloodV 63, p 260; Torok-Storb et al. (1982) Nature V 298, p 473; Mangan, etal. Blood V 63, p 260); and third, NK-like cells with the have beenisolated from patients with aplastic anemia (Mangan, et al. (1982) J.Clin. Invest. V 70, p 1148; and Nogasawa et al. (1981) Blood V 57, p1025). Moreover, recent studies in the mouse indicate that NK cells mayfunction to suppress hemopoiesis in vivo (Holmberg et al. (1984) J.Immunol V 133, p 2933). However, further investigation is desirablebefore the connection between NK activity and bone marrow dysfunction isconsidered conclusive.

Generally, when the dipeptide alkyl esters of the present invention areadministered to animals, an effective level is between about 1×10⁻⁴moles/kg and about 1×10⁻² moles/kg.

The following Examples are presented to more fully illustrate preferredembodiments of the present invention and are not intended to limit theinvention unless otherwise so stated in the accompanying claims.

EXAMPLE 1 Cell Preparations and Assays

PBM were separated from heparinized venous blood of healthy donors bycentrifugation over sodium diatrizoate-Ficoll gradients (Isolymph,Gallard-Schlesinger Chemical Mfg. Corp., Carle Place N.Y.).Monocyte-enriched populations ((MP) were prepared from glass adherentcells and MP-depleted lymphocytes from the nonadherent cells remainingafter incubation in glass Petri dishes and passage through nylon woolcolumns as detailed in Rosenberg et al. (1975) (J. Immunol V 122, pp926-831). PMN were collected by resuspending peripheral blood cells thatpenetrated sodium diatrizoate-Ficoll gradients and removing erythrocytesnu dextran sedimentation and hypotonic lysis as previously outlined(Thiele et al. (1985) J. Immunol. V 134, pp 786-793.

All cell exposures to the amino acids, dipeptides or their methyl esterswere carried out by suspending cells in Dulbecco's phosphate bufferedsaline (PBS) and incubating them at room temperature with the reagent atthe indicated concentration and time interval. After incubation, thecells were washed twice with Hanks' balanced salt solution andresuspended in medium RPMI 1640 (Inland Laboratories, Fort Worth, Tex.)supplemented with 10% fetal bovine serum (Microbiological Associates,Walkersville, Minn.) for assay of function.

Natural killing against K562 target cells was assessed by a 3 hour ⁵¹ Crrelease assay and percent specific lysis calculated as previouslydescribed (Thiele et al. (1985) J. Immunol. V 134, pp 786-793). Percentof control cytotoxicity was calculated using the formula: ##EQU1##

EXAMPLE 2 General Procedures for Generation, Purification andCharacterization of L-leucine Methyl Ester and Its Metabolites

MP or PMN (prepared as in Example 1) at a concentration of 25×10⁶ per mlwere suspended in PBS and incubated with 25 mM Leu-OMe for 20 minutes at22° C. Cell suspensions were then centrifuged at 1000 g for 10 minutesand the supernatants harvested and freeze-dried at -70° C., 100millitorr atmospheric pressure. In some experiments, Leu-OMe-treated MPor PMN were sonicated to increase the yield of the reaction product.Samples were than extracted with methanol for application to thin layerchromatography (TLC) plates (200 micromolar×20 cm², Analtech, Newark,Del.). Following development with chloroform/methanol/acetic acid(19:1:12.5 by volume), 1 cm bands were eluted with methanol, dried undernitrogen, and resuspended in 1 ml PBS. Mass spectra were obtained with aFinnegan Model 4021 automated EI/CI, GC/MS system coupled to an Incosdata system. Methane was used as the reagent gas for chemical ionization(CI) mass spectral analysis.

EXAMPLE 3 Lysosomotropic Substances and Formation of NK-toxic Products

The addition of Leu-OMe to human PBM was shown to cause rapid death ofMP and NK cells but not T or B lymphocytes (Thiele et al. (1985) J.Immunol. V 134, pp 786-793; Thiele et al. (1983) J. Immunol. V 131, pp2282-2290). Amino acid methyl esters are known to be lysosomotropiccompounds, and in previous studies it was found that the lysosomalinhibitors, chloroquine and NH₄ Cl, prevented Leu-OMe-induced MPtoxicity. To assess whether these agents similarly prevented formationof any NK toxic products, the following experiments were carried out,and the results shown in FIG. 1.

PBM (prepared as in Example 1) were incubated with various potential NKtoxic agents in the presence or absence of various lysosomal inhibitorsfor 40 minutes, washed to remove the inhibitor, incubated for 18 hoursto permit recovery from any transient inhibition caused bylysosomotropic agents and then tested for NK activity. As can be seen inFIG. 1, neither chloroquine, NH₄ Cl, nor Ile-OMe had any substantialpermanent effect on NK function. In contrast, 5 mM Leu-OMe ablated allNK activity. This activity of Leu-OMe was largely prevented bychloroquine, NH₄ Cl, or Ile-OMe. The products generated by MP or PMN,after exposure to Leu-OMe also completely removed all NK activity fromPBM. In contrast to the effect noted with Leu-OMe, the lysosomalinhibitors did not protect NK cells from the action of this product(s).Additional experiments indicated that the sonicates of MP or PMN had noeffect on NK function in this system whereas the supernatants orsonicates of Leu-OMe treated PMN or MP also depleted NK cells from MPdepleted lymphocytes.

These results therefore suggest that interaction of Leu-OMe with thelysosomal compartment of NP or PMN produced a product which was directlytoxic to NK cells through a mechanism that was no longer dependent onlysosomal processing within the NK cell or an additional cell type.

More particularly, the conditions of the manipulations leading to theresults shown in FIG. 1 were as follows:

Inhibitors of lysosomal enzyme function prevent generation of an NKtoxic product. PBM (5×10⁶ /ml) or PMN (25×10⁶ /ml) preincubated with 25mM Leu-OMe for 30 minutes were added to cells to be ablated. Cells wereincubated with these agents for another 30 minutes at 22° C., thenwashed and cultured for 18 hours at 37° C. Before assay of the abilityto lyse K562 cells. Data are expressed as percentage of controlcytotoxicity observed with an effector:target ratio of 40:1 (results atother E:T were similar).

EXAMPLE 4 Ablation of NK Function by PMN produced Leu OMe Product

When the NK toxic properties of MP-Leu-OMe, or PMN-Leu-OMe incubationmixtures were evaluated, it was found that his activity was stable inaqueous solutions for more than 48 hours at 4° C., but labile at 100°C., retarded on Sephadex G-10 columns; dialyzable through 1000 MWCO(molecular weight cut-off) membranes, and could be extracted bychloroform-methanol (3:1, by volume). As shown in FIG. 2, when ¹⁴C-leucine methyl ester was incubated with PMN and the supernatantssubsequently separated by TLC, three major peaks of ¹⁴ C activity werefound. One of these peaks corresponding to leucine methyl ester itselfand one to free leucine while the third represented a new product. Thisthird peak accounted for about 10% of the total ¹⁴ C-labeled material.When MP-depleted lymphocytes were exposed to each TLC fraction, thethird peak was found to contain all NK toxic activity. This NK toxicactivity not only appeared to be ¹⁴ C labeled but was also ninhydrinpositive, suggesting that it was a metabolite which still retained anamino group as well as part of the carbon structure of Leu-OMe. Anidentical ¹⁴ C labeled ninhydrin positive product was detected by TLC ofMP-Leu-OMe incubation mixture supernatants or sonicates. The productionby PMN or MP of this metabolite was inhibited by chloroaquine, NH₄ Cl,or Ile-OMe (data not shown).

Ablation of NK function is mediated by a metabolite of Leu-OMe. PMN(25×10⁶ /ml) were incubated with 25 mM ¹⁴ C-Leu-OMe for 30 minutes andsupernatants harvested for TLC analysis. MP-depleted lymphocytes(2.5×10⁶ cells/ml) were exposed to varying dilutions of each TLCfraction for 30 minutes, washed and cultured for 2 hours prior tocytotoxicity assay at E:T ratio of 20:1. Samples were considered tocontain an NK toxic product when percent specific lysis was less than25% of control. FIG. 2 shows these results.

EXAMPLE 5 Characterization Of The NK-toxic Metabolite

The nature of the new TLC peak found as described in Example 4 wasexamined by mass spectroscopy. As shown in FIG. 3A, when theTLC-purified, NK-toxic fraction was subjected to more spectral analysis,results showing peaks at M/Z 259 (MN+), 287 (M+C₂ H₅ +) and 299 (M+C₃ H₅+) indicated the presence of a compound of molecular weight 258. Thepresence of peaks at M/Z 244 (M⁺ --CH₃) and 272 (M+C₂ H₅ 5⁺ -CH₃)suggested that this compound contained a methyl ester group.Furthermore, the persistence of peaks corresponding to leucine (MN⁺=131, M+C₂ H₅ =159) and leucine methyl ester (MH⁺ =146, M+C₂ H₅ ⁺ =174)in spite of careful TLC purification of the NK toxic product from anyfree leucine or Leu-OMe present in the crude supernatants of theincubation mixtures suggested that a condensation product of Leu-OMesuch as Leu-Leu-OMe (MW258) was present in the NK toxic fractionisolated after incubation of PMN or MP with Leu-OMe.

When Leu-Leu-OMe was synthesized from reagent grade Leu-Leu, byincubation in methanol hydrochloride, it was found to have TLC mobilityidentical to NK toxic fractions of MP-Leu-OMe or PMN-Leu-OMe incubationmixtures. Furthermore, its CI mass spectrum as shown in FIG. 3B wasidentical to that of the 258 molecular weight compound found in theseincubation fractions.

Experiments further confirmed that Leu-Leu-OMe was the product generatedby MP or PMN from Leu-OMe that was responsible for the selectiveablation of NK function from human lymphocytes. Leu-Leu-OMe wassynthesized by addition of Leu-Leu to methanolic HCl. TLC analysisrevealed less than 2% contamination of this preparation with leucine,Leu-Leu, or leu-OMe, and CI mass spectral analysis (FIG. 3B) revealed nocontaminants of other molecular weights.

FIG. 3A shows the chemical-ionization CI mass spectra of TLC fractionswith NK toxic activity as described in FIG. 2, and also of Leu-Leu-OMesynthesized from reagent grade Leu-Leu (FIG. 3B).

EXAMPLE 6

In the representative experiments shown in FIG. 4, MP-depletedlymphocytes were exposed to varying concentrations of Leu-Leu-OMe for 15minutes at room temperature, then washed and assayed for ability to lyseK562 cells. No NK function could be detected in lymphocyte populationsexposed to greater than 50 micromolar Leu-Leu-OMe. As previouslydemonstrated (Thiele et al. (1983) J. Immunol. V 131 pp 2282-2300),exposure of such MP-depleted lymphocyte populations to 100 fold greaterconcentration of leucine or leu-OMe had no irreversible effect on NKfunction. Leu-Leu or the D-stereoisomer, D-Leu-D-Leu-OMe, also had noinhibitory effect. While Leu-Leu-Leu-OMe caused dose-dependent loss ofNK function, 5-fold greater concentrations of this tripeptide methylester were required to cause an effect equivalent to that of thedipeptide methyl ester of L-leucine. When lymphocyte populations exposedto varying concentrations of Leu-Leu-OMe were further analyzed, it wasfound that exposure to more than 50 micromolar Leu-Leu-OMe resulted inthe loss of K562 target binding as well as complete depletion of cellsstained by Leu 11b, and anti-NK cell monoclonal antibody (data notshown). Thus, the MP- or PMN-generated product of Leu-OMe which isdirectly toxic for human NK cells is the dipeptide condensation productLeu-Leu-OMe.

The condition of the manipulations resulting in the data leading to FIG.4 are further detailed as follows: for loss of NK function afterexposure to Leu-Leu-OMe, MP-depleted lymphocytes (2.5×10⁶ cells/ml) wereincubated for 15 minutes with the indicated concentrations of leucinecontaining compounds. Cells were than washed, cultured at 37° C. for 2hours (Expt. 1) or 18 hours (Expt. 2) and then assayed for NK activity.Results are given for E:T ratio of 20:1.

EXAMPLE 7 NK Ablation by a Variety of Dipeptide Methyl Esters

In previously reported studies, Leu-OMe was unique among a wide varietyof amino acid methyl esters in its ability to cause MP or PMN dependentablation of NK cell function from human PBM (Thiele et al. (1985) J.Immunol. V 134, pp 786-793). The identification of Leu-Leu-OMe as theMP-generated metabolite responsible for this phenomenon suggested thateither MP/PMN did not generate the corresponding dipeptide methyl estersin toxic amounts from other amino acids, or that Leu-Leu-OMe was uniqueamong dipeptide methyl esters in its toxicity for NK cells. Therefore,experiments were carried out to assess the effect of other dipeptidemethyl esters on NK cell function. The methyl esters of a variety ofdipeptides were synthesized and analyzed for the capacity to deplete NKcell function. Each dipeptide methyl ester was assessed in a minimum ofthree experiments. As is shown by the results displayed in FIG. 5,Leu-Leu-OMe is not the only dipeptide methyl ester which exhibits NKtoxicity. When amino acids with hydrophobic side chains were substitutedfor leucine in either position, the resulting dipeptide methyl estergenerally displayed at least some degree of NK toxicity. In particular,Leu-Phe-OMe, Phe-Leu-OMe, Val-Phe-OMe, and Val-Leu-OMe producedconcentration-dependent ablation of NK function at concentrationscomparable to those at which Leu-Leu-OMe was active. The sequence ofactive amino acids was important, however, as evidenced by the findingthat Phe-Val-OMe was markedly less active than Val-Phe-OMe. Similarly,Leu-Ala-OMe was NK inhibiting, whereas 10-fold greater concentrations ofAla-Leu-OMe had no NK inhibitory effects. Furthermore, Phe-Phe-OMe wasless NK toxic than either Leu-Phe-OMe or Phe-Leu-OMe and Val-Val-OMe wasless active than either Leu-Val-OMe or Val-Leu-OMe, yet Val-Phe-OMe wasamong the most potent of the NK toxic dipeptide methyl esters. Thus,conformational aspects of the dipeptide methyl ester amino acid sidechain also seem to be of importance in producing the different levels ofobserved NK toxicity.

When amino acids with hydrophilic, charged or hydrogen side chains weresubstituted for leucine, the resulting dipeptide methyl esters eitherhad greatly reduced NK toxicity, as in the case of Gly-Leu-OMe orLeu-Gly-OMe, or no observed NK inhibitory effects, as in the case ofLeu-Arg-OMe, Leu-Tyr-OMe, Ser-Leu-OMe, Lys-Leu-OMe or Asp-Phe-OMe.Furthermore, when the D-stereoisomer was present in either position of adipeptide methyl ester, no toxicity was observed for NK cells (FIG. 5).When unesterified dipeptides were assessed for their effect on NKfunction, as in the case of Leu-Leu (FIG. 4), up to 5×10⁻³ Mconcentrations of Leu-Phe, Phe-Leu, Val-Leu, and Val-Phe had no effecton NK cell survival or lytic activity (data not shown).

D-Leu-D-Leu-OMe had no effect on Leu-Leu-OMe mediated NK toxicityalthough high levels of zinc appeared to inhibit this Leu-Leu-OMetoxicity.

Previous experiments had demonstrated that compounds such as Val-OMe,Phe-OMe, or combinations of Val-OMe and Phe-OMe did not delete NKfunction from human PBM (Thiele et al. (1985) J. Immunol. V 134, pp786-793), despite the current finding that dipeptide methyl esterscontaining these amino acids were potent NK toxins. In order todetermine whether MP or PMN could generate the relevant dipeptide methylesters from these amino acid methyl esters, TLC analysis of thesupernatants of MP and PMN incubated with these compounds was carriedout. It was found that MP and PMN did generate detectable amounts ofdipeptide methyl esters from these L-amino acid methyl esters. However,when equal concentrations of Leu-OMe, Val-OMe, or Phe-OMe were added toMP or PMN, the concentrations of Val-Val-OMe generated were 50 to 80% ofthose found for Leu-Leu-OMe, while Phe-Phe-OMe was detected at only10-30% of the levels of Leu-Leu-OMe. Dipeptide methyl esters were notgenerated from D-amino acid methyl esters.

FIG. 5 shows the NK toxicity of dipeptide methyl esters. MP-depletedlymphocytes were treated with varying concentrations of dipeptide methylesters as outlined in FIG. 4. Results are give for the mean ±SEM of atleast 3 separate experiments with each compound.

EXAMPLE 8 NK Toxicity of an Artificially Hydrophobic Dipeptide MethylEster

Beta methyl aspartyl pheylanaline was prepared by methanolichydrochloride methylation of aspartyl phenylalanine methyl ester. The NKtoxicity of both aspartyl phenylalanine methyl ester and beta methylaspartyl phenylalanine methyl ester was measured as described for thedipeptide methyl esters in Example 7. As the data in Table 2 indicates,when the polar side chain of the aspartyl amino acid dipeptide componentis esterified with a methyl group, this being a conversion from relativehydrophilicity to substantial hydrophobicity, NK toxicity becomesapparent. Although yet not as toxically effective as a number of thehydrophobic-type dipeptides in Example 7, the data in Table 2 indicatethat a dipeptide methyl ester comprising synthetic hydrophobic(lipophilic) amino acids may be used to inhibit NK function.

                  TABLE 2                                                         ______________________________________                                        L-ASPARTYL (beta-METHYL ESTER)-L-                                             PHENYLALANINE METHYL ESTER IS NK TOXIC                                        WHlLE L-ASPARTYL-L-PHENYLALANlNE METHYL                                       ESTER IS NOT                                                                                      NK Function                                                                   % Specific                                                Preincubation       Cytotoxicity                                              ______________________________________                                        Nil                 50.8                                                      Asp--Phe--OMe:                                                                 100 micromolar     54.2                                                       250 micromolar     45.7                                                       500 micromolar     45.7                                                      1000 micromolar     46.9                                                      Asp-(beta-OMe)--Phe--OMe:                                                      100 micromolar     38.9                                                       250 micromolar     13.9                                                       500 micromolar     2.8                                                       1000 micromolar     -0.1                                                      ______________________________________                                    

EXAMPLE 9 In Vivo Effects on Cytotoxic Cell Function

Leu-Leu-OMe or Leu-Phe-OMe were suspended in PBS, pH 7.4. Thenindividual C3H/HeJ mice (25 gram size) were administered by tail-veininjection either 2.5×10⁻⁵ moles (6.5 mg) of Leu-Leu-OMe, 2.5×10⁻⁻⁵ moles(7.1 mg) Leu-Phe-OMe, or an equal volume of the PBS diluent, this dosebeing about 1×10⁻³ moles per kg. For 15-30 minutes post-injection,Leu-Leu OMe and Leu-Phe OMe-treated animals but not the control animalsexhibited decreased activity and an apparent increase in sleep.Subsequent to this quiescent period no difference in activity orappearance in the mice was noted. Two hours post-injection, the micewere sacrificed and their spleen cells were assayed for NK function in astandard 4 hour assay against YAC-1 tumor targets. In all mice, totalcell recovery ranged from 1×10⁸ to 1.1×10⁸ spleen cells per animal. Asnoted in Table 3, the control mouse spleen cells exhibited greaterkilling at 25:1 and 50:1 effector to target cell ratios than did thespleen cells of treated mice at 100:1 and 200:1 E/T, respectively. Thus,Leu-Leu-OMe or Leu-Phe-OMe caused a greater than 75% decrease in spleniclytic activity against YAC-1 tumor targets.

                  TABLE 3                                                         ______________________________________                                        Cytotoxic Cell Function                                                                   Effector:Target Ratio                                                         25:1 50:1      100:1   200:1                                                  Percent lysis of target cells                                     ______________________________________                                        Control       8.29   12.88     20.60 29.29                                    Leu--Leu--OMe 2.37   4.58      7.12  12.77                                    Leu--Phe--OMe 3.89   4.68      6.91  11.91                                    ______________________________________                                    

EXAMPLE 10 Differential Sensitivity of Natural Killer Cells (NK) andMononuclear Phagocytes (MP) to Leucylleucine-Methyl Ester (Leu-Leu-OMe)

In the experiments depicted in FIG. 6, freshly isolated PBM (2.5×10⁶ /mlPBS and 1 g/l glucose) were incubated at room temperature with varyingconcentrations of Leu-Leu-OMe. After a 15 minute exposure to thiscompound, the cells were washed, incubated for 2 hours at 37° C. andthen assessed for the percentage of remaining viable cells which werestained by anti-MP or anti-NK monoclonal antibodies. Preincubation withgreater than 25-50 micromolar Leu-Leu-OMe led to loss of NK cells. Thisconcentration of Leu-Leu-OMe did not deplete MP from PBM but higherconcentrations of Leu-Leu-OMe caused loss of MP. The data is FIG. 6 showthese results.

Anti-MP monoclonal antibodies (63D3) and anti-NK monoclonal antibodies(leu 11b) were obtained from Becton Dickinson Monoclonal Center, Inc.,Mountain View, Calif. The antibody staining and Fluorescence ActivatedCell Sorter (FACS) procedure was that of Rosenberg et al. (1981) (J.Immunol. V 126, p 1473). Data are expressed as percent of antibodystaining in control cells (mean ±SEM, n=4).

EXAMPLE 11 Effects of Leu-Leu-OMe on a Variety of Cell Types

While it was clear that a substantial percentage of lymphocytes remainedviable following exposure to even 1 mM Leu-Leu-OMe, the finding thatdisparate cell types such as MP and NK were both susceptible toLeu-Leu-OMe mediated toxicity raised the possibility that this agent wasa non-specific cell toxin. Therefore, the series of experiments depictedin FIG. 7 was performed to assess other cell types for evidence oftoxicity following exposure to Leu-Leu-OMe.

To facilitate screening of multiple cell types for evidence of celldeath following exposure to Leu-Leu-OMe, a ⁵¹ Cr release assay wasdevised. In preliminary experiments it was noted that ⁵¹ Cr release fromMP-enriched populations exposed to varying concentrations of Leu-Leu-OMecorrelated very closely with concentration-dependent loss of anti-MPantibody staining cells from PBM after similar incubation. Followingbrief exposures to Leu-Leu-OMe at room temperature, the loss of anti-MPantibody staining cells from PBM or the release of ⁵¹ Cr fromMP-enriched populations was always detectable within a 30 to 60 minuteperiod of culture at 37° C. and maximal effects were seen within 3 to 4hours.

Therefore, ⁵¹ Cr release in a 4 hour assay was used in these experimentsto assess toxicity from Leu-Leu-OMe. As shown in the first graph of FIG.7, when the whole PBM population was exposed to varying concentrationsof Leu-Leu-OMe, detectable ⁵¹ Cr release was observed after exposure to25 to 50 micromolar Leu-Leu-OMe, but only upon exposure to greater than100 micromolar Leu-Leu-OMe was the maximal achievable ⁵¹ Cr release fromPBM observed. When MP-enriched adherent cells (AC) were similarlyassessed, minimal ⁵¹ Cr release was observed after exposure to 25-50micromolar Leu-Leu-OMe whereas upon incubation with higherconcentrations of this agent, more ⁵¹ Cr release from AC was observedthan with PBM. When nylon wool non-adherent lymphocytes (NAC) wereassessed, small but significant ⁵¹ Cr release was observed with 25 to 50micromolar Leu-Leu-OMe. When NAC were exposed to increasingconcentrations of Leu-Leu-OMe, greater quantities of ⁵¹ Cr release wereobserved. N-SRBC positive cells showed a dose-dependent Leu-Leu-OMeinduced ⁵¹ Cr release pattern indistinguishable from that of NAC. Sinceboth antibody staining (FIG. 6) and functional studies (FIG. 4) haveshown that 100 micromolar Leu-Leu-OMe causes maximal depletion of NK,this finding suggested that other lymphocytes were also susceptible toLeu-Leu-OMe toxicity at concentrations greater than 100 micromolar. WhenT4 enriched populations of T cells were assessed, however, it was clearthat even 1000 micromolar Leu-Leu-OMe caused minimal ⁵¹ Cr release fromthis population. In contrast, when N-SRBC positive cells were depletedof OKT4 positive cells, the remaining T8-enriched population producedhigh levels of ⁵¹ Cr release following exposure to Leu-Leu-OMe.

When cell lines of myeloid or lymphoid origin were similarly assessed,selective toxicity of Leu-Leu-OMe was again observed. The human T cellleukemia line MoLT-4 demonstrated no detectable Leu-Leu-OMe toxicityover a broad concentration range. The human plasma cell lines HS-Sultanand the B lymphoblastoid line Daudi demonstrated no significant ⁵¹ Crrelease or alteration in subsequent proliferative rate (data not shown)after exposure to a broad range of Leu-Leu-OMe concentrations. When thesusceptibility of EBV-transformed B cell lines or clones to this agentwas assessed, no significant toxicity of less than 250 micromolarLeu-Leu-OMe was seen. However, with higher concentrations ofLeu-Leu-OMe, a variable degree of toxicity was seen. Some EBV linesconsistently displayed less than 20% ⁵¹ Cr release even after exposureto 1 mM Leu-Leu-OMe, while other lines produced 25-35% ⁵¹ Cr releaseafter exposure to 250 micromolar Leu-Leu-OMe. In contrast, the humancell line U937 was susceptible to concentration-dependent Leu-Leu-OMetoxicity in a pattern indistinguishable from that of the peripheralblood MP with which this cell line shares many phenotypic and functionalcharacteristics. After exposure to more than 250 micromolar Leu-Leu-OMe,extensive ⁵¹ Cr release was observed and no viable proliferating U937cell could be detected (data not shown). Similarly, the erythroleukemialine K562 demonstrated no significant ⁵¹ Cr release or alteration insubsequent proliferative rate (date not shown) upon exposure to 100micromolar or lower concentrations of Leu-Leu-OMe. With higherconcentrations of Leu-Leu-OMe, modest amounts of ⁵¹ Cr release andpartial loss of proliferative capacity were observed (data not shown).In contrast, a variety of cell types of non-lymphoid, non-myeloid originincluding human umbilical vein endothelial cells, the human renal cellcarcinoma line, Currie, the human epidermal carcinoma line, HEp-2, andhuman dermal fibroblasts demonstrated no significant Leu-Leu-OMe induced⁵¹ Cr release. Furthermore, incubation of each of these non-lymphoidcell types with 500 micromolar Leu-Leu-OMe had no discernible effect onsubsequent proliferative capacity (data not shown).

HS-Sultan, a human plasma cell line (Goldblum, et al. (1973) Proc.Seventh Leucocyte Culture Conference, ed by blastoid cell line (Klein etal. (1968) Cancer Res. V 28, p 1300), MoLT-4, an acute lymphoblasticT-cell leukemia line (Monowada et al. (1972) J. Nat'l. Canc. Inst. V 49,p 891), and U-937, a human monocyte-like cell line (Koren et al. (1979)Nature V 279, p 891) were obtained from the American Type CultureCollection, Rockville, Md. These lines as well as HEp-2 a humanepidermoid carcinoma line (a generous gift of Dr. R. Sontheimer,UTHSCD); Currie, a human renal cell carcinoma line (a generous gift ofDr. M. Prager, UTHSCD); and K562, a human erythroleukemia line (agenerous gift of Dr. M. Bennett, UTHSCD) were maintained in culture inmedium RMPI supplemented with 10% FBS. Human dermal fibroblasts (agenerous gift of Dr. T. Geppert, UTHSCD) were serially passaged inculture as well while human umbilical vein endothelial cells (a generousgift of Dr. A. Johnson, UTHSCD) were used after one subculture. EpsteinBarr virus (EBV) transformed B lymphoblastoid cell lines JM.6 and SM.4(kindly provided by Dr. J. Moreno, UTHSCD) and cloned CBV transformed Bcell lines SDL-G2 and D8-219 (a generous gift of Drs. L. Stein and M.Dosch, Hospital for Sick Children, Toronto, Canada) were maintained inculture in medium RPMI supplemented with 10% FBS.

In some experiments, toxicity of Leu-Leu-OMe for a variety of cellpopulations was assessed by ⁵¹ Cr release. In assays where cellsobtained from suspension culture were to be used, cells were labeledwith Na₂ ⁵¹ CrO₄ (ICN, Plainview, N.Y.) for 60-90 minutes at 37° C. andthen washed three times. Cells were then suspended in PBS (2.5×10⁶ /ml)and incubated in microtiter plates, 50 microL/well with indicatedconcentrations of Leu-Leu-OMe for 15 minutes at room temperature. Inassays where cells were obtained from monolayer cultures, microtiterwells were seeded with cells (5×10⁴ /well) and cultured for 24 hours at37° C. Cells were then labeled with Na₂ ⁵¹ CrO₄ while in adherentculture. Following ⁵¹ Cr labeling, wells were thoroughly washed andvarying concentrations of Leu-Leu-OMe added in 50 microL PBS and theplates incubated for 15 minutes at room temperature.

Following such initial serum-free incubations, 200 microL/well of mediumRPMI containing 10% FBS were added and the plates incubated for another4 hours prior to removal and 100 microliters of supernatant.Radioactivity in the supernatant was measured in an auto-gammascintillation spectrometer (Packard Instrument Co., Downers Grove,Ill.). The percent specific release was calculated from the formula:##EQU2## in which maximal release refers to cpm obtained in wellscontaining 50% lysing agent (American Scientific Products, McGraw Park,Ill.) and spontaneous release refers to cpm released by cells incubatedin control medium in the absence of Leu-Leu-OMe or the lysing agent.Only experiments in which spontaneous release was 25% were used forsubsequent data interpretation.

While the MP-like tumor line U937 was virtually identical to MP insusceptibility to Leu-Leu-OMe, none of the non-lymphoid, non-myeloidcell lines tested demonstrated such susceptibility to Leu-Leu-OMemediated toxicity.

The current example demonstrates that at concentrations 10 to 20 foldgreater than those at which cytotoxic cells are ablated, Leu-Leu-OMedoes have some minimal toxicity for certain non-cytotoxic lymphoid cellssuch as EBV transformed B cells and K562 cells. Yet, while it isimpossible to exhaustively exclude the possibility that certainnon-cytotoxic cells might also be equally sensitive toLeu-Leu-OMe-mediated toxicity, at present the ability to function as amediator of cell mediated cytotoxicity is the one unifyingcharacteristic of the cell types which are rapidly killed by exposure toLeu-Leu-OMe.

In developing the data expressed in FIG. 7, cells (2.5×10⁶ /ml) wereexposed to the indicated concentrations of Leu-Leu-OMe for 15 minutes atroom temperature, then specific ⁵¹ Cr release during the next four hourswas assessed. Data for the EBV transformed lines JM.6, SDL-G2, D8-219,and SM.4, respectively, are shown in order from top to bottom.

EXAMPLE 12 Relative Sensitivity of CTL and NK to Leu-Leu-OMe

Experiments were also designed to assess the relative sensitivity of NKand CTL to Leu-Leu-OMe. In the studies detailed in FIGS. 8 and 9,cytotoxicity assays were performed over a broad range of E:T ratios andunits of lytic activity arising from equal numbers of respondinglymphocytes were calculated and compared. As shown in FIG. 8, bothspontaneous NK and precursors of activated NK were totally eliminated byexposure to 100 micromolar Leu-Leu-OMe while CTL precursors, thoughdiminished, were generally still present at greater than 50% of controllevels. Only after exposure to greater than 250 micromolar Leu-Leu-OMewere all CTL precursors eliminated.

FIG. 8 shows that incubation with Leu-Leu-OMe eliminates precursors ofcytotoxic T lymphocytes (CTL) and activated NK-like cells (AcNK).Non-adherent lymphocytes (2.5×10⁶ /ml) were incubated with the indicatedconcentrations of Leu-Leu-OMe for 15 minutes. Cells were then washed andeither placed in mixed lymphocyte culture or assayed for specific lysisof K562 cells (NK). After 6 day MLC, cells were assayed for specificlysis of allogeneic stimulator lymphoblasts (CTL) or K562 (AcNK). Dataare expressed as percent of control lytic units (mean+SEM, n=6).

When the elimination of CTL and activated NK precursors by Leu-Leu-OMewas compared to that of spontaneous NK, the mean Leu-Leu-OMeconcentration required to diminish lytic activity by 75% wassignificantly greater for elimination of CTL precursors (123±25micromolar) than for elimination of precursors of activated NK (50±5micromolar, p 0.05). Both values were also higher than the meanconcentration of Leu-Leu-OMe required to diminish spontaneous NK lyticactivity by 75% (35 micromolar±4 micromolar). FIG. 9 shows that,following activation, CTL and AcNK became identical in sensitivity toLeu-Leu-OMe. After 6 day MLC, cells were incubated for 15 minutes withthe indicated concentrations of Leu-Leu-OMe, then assayed with CTL orAcNK activity as for FIG. 8. Thus, only after MLC activation did CTLdisplay a sensitivity to Leu-Leu-OMe toxicity that was equal to that ofNK cells.

Changes may be made in the construction, operation and arrangement ofthe various elements, steps and procedures described herein withoutdeparting from the concept and scope of the invention as defined in thefollowing claims.

What is claimed is:
 1. A method of inhibiting the natural killer cell orcytotoxic T-lymphocyte mediated rejection of tissue transplanted into ahost, the method comprising treating said host with a therapeuticallyeffective amount of an alkyl ester of a dipeptide consisting essentiallyof the L-amino acids leucine, phenylalanine, valine, isoleucine,alanine, proline, glycine, or aspartic acid beta methyl ester,individually or in combination.
 2. A method of inhibiting the naturalkiller cell or cytotoxic T-lymphocyte mediated rejection of tissuetransplanted into a host, the method comprising treating said host witha therapeutically effective amount of an alkyl ester of a dipeptideconsisting essentially of natural or synthetic L-amino acids withhydrophobic side chains, said amount being sufficient to deactivatenatural killer cells.
 3. The method of claim 2 wherein dipeptide isL-leucyl L-leucine, L-leucyl L-phenylalanine, L-valyl L-phenylalanine,L-phenylalanyl L-leucine, L-leucyl L-isoleucine, L-phenylalanylL-phenylalanine, L-valyl L-leucine, L-leucyl L-alanine, L-valylL-valine, L-prolyl L-leucine, L-leucyl L-valine, L-phenylalanylL-valine, L-glycyl L-leucine, L-leucyl L-glycine, or L-aspartyl betamethyl ester L-phenylalanine.
 4. The method of claim 2 wherein thedipeptide is L-leucyl L-leucine, L-leucyl L-phenylalanine, L-valylL-phenylalanine, L-phenylalanyl L-leucine, L-leucyl L-isoleucine,L-phenylalanyl L-phenylalanine or L-valyl L-leucine.
 5. The method ofclaim 1 wherein the effective amount is between about 1×10⁻⁴ moles perkg and 1×10⁻² moles per kg.
 6. The method of claims 1 or 2 wherein thealkyl ester is a methyl ester, an ethyl ester, a propyl ester, anisopropyl ester, a butyl ester or an isobutyl ester.
 7. A method ofinhibiting cytotoxic T-lymphocyte-mediated rejection of tissuetransplanted into a host, the method comprising treating said host witha cytotoxic T-lymphocyte-inhibitory amount of a dipeptide alkyl esterconsisting essentially of an L-amino acid selected from the groupconsisting of leucine, phenylalanine, valine, isoleucine, alanine,proline, glycine, or aspartic acid beta methyl ester, individually or incombination.
 8. The method of claim 7 wherein the dipeptide alkyl esteris Leu-Leu-OMe.